The Folding Transition State of the Cold Shock Protein is Strongly Polarized

The cold shock protein CspB from Bacillus subtilis consists of a three-stranded (β1–β3) and a two stranded (β4–β5) sheet, which form a closed β barrel structure. CspB folds and unfolds rapidly in a two-state reaction, and the unfolded and the folded molecules interconvert with a time constant of 30 ms at the midpoint of the urea-induced transition (at 25 °C). The transition state of folding is native-like, as judged by the Tanford βT value of ≥0.9. By using a mutational approach and Φ value analysis, we find that the folding transition state of CspB is energetically polarized. Despite the high βT value, most Φ values are low. Values close to 1 were found for only a few residues, particularly in strand β1 (Lys5, Val6, Lys7, Asn10). The interactions of the Asn10 side-chain with the backbone at positions 12 and 13 define the turn that connects the strands β1 and β2. Lys5 and Val6 in β1 interact with residues in β4, and their high Φ values indicate that an energetic linkage between β1 and β4 and thus between the two sheets exists already in the transition state. We compared our experimental Φ values with theoretical predictions of the folding pathway of cold shock proteins. Several of them suggest that the entire first sheet is formed in the transition state, and some identify the β1–β4 pairing as a crucial step in folding. Alternative paths that involve formation of the second sheet and β3–β5 pairing reactions were, however, suggested as well. The calculations gave coarse-grained pictures that are limited in resolution to the two sheets of CspB or to the elements of secondary structure. They did not identify the key residues with the high Φ values within these structural elements.